Methods for preparing and using clonogenic fibroblasts and transfected clonogenic fibroblasts

ABSTRACT

The invention relates to a process for preparing clonogenic fibroblasts, with tissue being removed from the donor and the individual cells being isolated from the tissue, the resulting cell suspension being strained, the cells which are contained in the cell suspension being washed and the cells being converted into a tissue culture, with the exception of the isolation of individual cells by mechanical comminution, followed by an enzymic treatment with collagenase alone, and with at least one gene being inserted into the fibroblasts by means of the transfection, which gene encodes a biologically active protein, preferably a therapeutically active protein, for example a growth factor, a hormone, an enzyme, a coagulation factor or a coagulation inhibitor.

The present invention relates to a process for preparing clonogenicfibroblasts, to processes for genetically transfecting fibroblasts andto genetically transfected fibroblasts which are thus obtained.

In the therapy of a wide variety of diseases, it is desirable to supplyparticular biologically active molecules, which can also be produced bythe human body, to the body in an increased, medicinally active dose.However, the medicinal supply of biologically active molecules whichhave been prepared outside the body also has the disadvantage that suchactive compounds have to be administered parenterally frequently andoften even several times daily, with a single, subcutaneousadministration often not being sufficient and intravenous doses whichare administered several times daily being required instead.

SU 13 17 021 A1 relates to diploid stem cells from human skin and toembryonic muscle cells which have been isolated for the purpose ofcultivating viruses.

SU 15 18 370 A1 relates to embryonic stem cell cultures of human skinand muscles for the purpose of producing diagnostic preparations.

Chapter 9 of the handbook written by R. Jan Freshney, "Culture of AnimalCells", 2nd Edition, Alan R. Liss Inc., New York, 1988, describes theisolation of tissue and primary (cell) cultures. On the following pages,a process for preparing human fibroblasts is described in which thetissue has been removed from the skin of donors within the context of abiopsy, whereupon the individual cells are isolated from the tissue, thecells which are present in the cell suspension are washed and the cellsare converted into a tissue culture. However, this process comprises,exclusively within the context of isolating cells, a mechanicalisolation of cells followed by an enzymic treatment with collagenase.

An object of the present invention is to provide a process by whichcells of this nature are made available which have been altered in sucha way that they are able to produce biologically active molecules.

The present invention discloses a process for preparing human clonogenicfibroblasts and also a process for genetically transfecting fibroblasts.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the mean doubling time of cell cultures prepared fromvarious donors. The numbers behind the curves indicate the age of therespective donors.

FIGS. 2A-2F depict a comparison of proliferation of fibroblasts as afunction of the manner in which the cells were prepared. The numbers inthe lower light hand corners indicate the age of the donor.

FIGS. 3A (skin) and 3B (serosa) depict the proliferation of autologousfibroblasts from peritoneal cells.

FIGS. 4A and 4B depict the growth of diploid fibroblasts in the presenceand absence of feeder cells.

FIGS. 5A, SE, and 5C depict the amount of TNFα, GM-CSF and IL-6 detectedin the supernatants of irradiated and non-irradiated cell cultures.D=days; H=hours.

FIG. 6 depicts the structure of the retroviral vectors which were used.

FIG. 7 depicts GM-CSF production (ηg/24 hr) by N2/CMV-GM-CSF/CMS 5#6cells after irradiation with. 50 Gy. 10⁶ irradiated cells were plated onday 0 and the GM-CSF concentration in the 24 h culture supernatant wasdetermined in a bioassay.

FIG. 8 depicts production of GM-CSF from irradiated populations on days0, 5, and 7. The table gives the absolute value of the leucocytesubpopulations on treatment days 0, 5, and 7. Cyclo=cyclophosphamide,d0+d2 (i.p., 150 mg/kg). GM-CSF=cyclophosphamide, d0+d2 (i.p., 150mg/kg)+rmGM-CSF, d3-d10 (s.c., 100 ηg, 2×daily).CMS5/GM-CSF=cyclophosphamide, d0+d2 (i.p., 150mg/kg)+N2/CMV-GM-CSF/CMS5, d3 (s.c., 10⁷ cells).

FIG. 9 depicts the peripheral leucocyte counts incyclosphosphamide-treated BALB/c mice with and without treatment withmGM-CSF or GM-CSF secreting fibrosarcoma cells.

FIGS. 10A, 10B, 10C, 10C, and 10D depict the leucocyte count as numbersof cells per ηl as a function of time after inoculation withnon-irradiated CMS-5 cells transfected with pCMV.GCSF iresNEO (FIGS. 10Aand 10C) or pCMV.GCSF iresTK/NEO (FIG. 10B and 10D). FIGS. 10C and 10Ddepict the leucocyte counts following inoculations of tumor cells andgancyclovir. FIGS. 10E, 10G, 10F, and 10H depict the chronologicaldevelopment of tumor size in BALB/c mice after inoculation withnon-irradiated CMS-5 cells transfected with pCMV. GCSF. iresNeo (FIGS.10E and 10G) or pCMV. GCSF iresTK/Neo (FIGS. 10F and 10H).

FIGS. 11 A, B, C, and D depict the leukocyte numbers followingchemotherapy and cytokine therapy in the presence or absence oftransfected fibroblasts. FIG. 11A, Cycloposphamide. FIG. 11B,Cyclophosphamide+rhG-CSFbbid. FIG. 11C, Cyclophosphamide+G-CSF/BALB 3T3.FIG. 11D, Cyclophosphamide+irradiated G-CSF/BALB 3T3.

In the novel processes, tissue samples are first removed from the serosaand, in a preferred form, from the skin of the donor, with these sampleshaving a size of from 0.5 to about 2 cm². Tissue samples of this naturecan be removed using known, routine surgical biopsy methods.

The samples which are obtained within the context of the biopsy are thenchopped with a scalpel or a comparable instrument into pieces of lessthan 2 mm in diameter. These pieces can then be laid out on cell cultureplates with the epidermal layer upwards. In a preferred manner,Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calfserum and the customary additives can be used as the culture medium.

Within the context of the present invention, it has emerged that it ismore advantageous if the tissue samples are first of all only choppeddown to a size of about 0.5 cm² and these pieces are then incubated inculture medium (DMEM), with the medium containing enzymes which promoteisolation of the individual cells. Within the scope of the presentinvention, collagenase, dispase (a neutral protease) or hyaluronidaseare preferably employed as enzymes of this nature which promoteisolation of the individual cells, with it being possible to use theenzymes either individually or in different combinations.

The cells which have been liberated as a result of the enzymic treatmentare then washed with a phosphate-buffered solution of sodium chlorideand sown in cell culture bottles at a density of about 2×10 ⁴ /cm².

The fibroblast cultures are customarily provided with fresh growthmedium twice a week. When the cultures have reached confluence, thecells can be harvested by treating them with trypsin/EDTA. A new growthcycle begins after the cells have been washed, counted and sown onceagain at a density of 1×10⁴ cells/cm².

It has emerged that, within the context of the present invention, it isadvisable to add so-called feeder cells (about 5×10⁵ cells) to about 10²clonogenic fibroblast cells in dishes having a diameter of 10 cm wherebythe efficiency of plating can be improved to up to 9-24%. The feedercells are irradiated with an intensity of about 50 Gy or 100 Gy. In apreferred manner, human embryonic Ws-1 fibroblasts, which can beobtained from the American Type Culture Collection, or else mouse NIH3T3fibroblasts, are used for this purpose.

In a particularly effective process for preparing human clonogenicfibroblasts, the tissue samples which are obtained by biopsy are firstof all chopped into pieces which are not more than 0.5 cm² in size.These tissue pieces are then digested, at 4° C. for 16 hours, with theenzyme dispase at a concentration of 2.5 units/ml. After the epidermishas been removed, the skin cells are chopped into pieces which are onlya few millimeters in size. The material which has thus been obtained issubjected to further digestion, at 37° C. for 3 hours in a shaking waterbath, with a mixture of collagenase (200units/ml) and hyaluronidase(300units/ml). Finally, the cells are strained through a sieve having amesh size of 70 μm, washed and transferred to the cell culture.

It was established that it was possible to obtain about 10¹¹ cells,after an average time of 89 days (±8 days), from donors who were youngerthan 60 years old.

FIG. 1 shows that the mean doubling time is about 4.3 (±0.6) days.Further growth was markedly slower in some cases and in quite a numberof cases a plateau was reached which allowed no further increase in thecell count. However, in a quite a number of cultures, it was possible toreach a cell count of more than 10¹⁵ cells without any flattening of thecurve being observed. It was found that the cultures originating fromolder donors have slower growth rates and stagnate sooner.

FIG. 2 shows a comparison of fibroblasts as a function of the manner inwhich the cells were prepared. The enzymically prepared fibroblastsclearly exhibited a greater ability to proliferate than did those whichwere only obtained from skin biopsies by mechanical preparation. Thecell cultures originating from different donors always exhibitedsuperior multiplication behavior when the cells were prepared followingan enzymic treatment as compared with those cells which only grew out ofthe biopsy samples.

FIG. 3 shows that it was also possible to obtain autologous fibroblastseffectively from peritoneal cells using the enzymic preparation method.While serosa and skin cultures initially grew at equal rates, thediploid fibroblasts from serosa reached the plateau sooner than did skincultures of the same age.

The plating efficiency of the diploid fibroblasts is of great importancewith regard to permanently transfected clones. FIG. 4 shows that diploidfibroblasts only grew poorly in the absence of feeder cells. The platingefficiency was increased to from 9 to 24% by adding irradiated humanWS-1 fibroblasts. The supernatants from non-irradiated cells wereharvested after 3 and 8 days, as were those from cells which wereirradiated with 20 Gy and 100 Gy. From 1.2 ng to 20.5 ng ofinterleukin-6 were found per 24 hours and 10⁶ cells in the supernatantsof non-irradiated diploid fibroblasts. The irradiation did not cause anysignificant change in interleukin-6 secretion. By contrast,non-irradiated diploid fibroblasts produced negligible quantities ofGM-CSF (granucyte macrophage colony stimulating factor) or TNFα (tumornecrosis factor), whereas up to 90 pg of GM-CSF and 50 pg of TNFα weremeasured per 24 hours and 10⁶ cells in the supernatants afterirradiation. The induction of these cytokines by irradiation wasstrongly dependent on the particular individual cultures.

The diploid fibroblasts which can be prepared by the novel process may,for example, be readily obtained from cancer patients. These clonogenicfibroblasts serve as a suitable starting material for the genetransfection. Transfected fibroblasts of this nature may then besupplied once again to the donors as autologous cells.

The fibroblasts which can be obtained by the process which has beendescribed in detail above may be genetically manipulated by means oftransfection. During the gene transfection, one or more genes encoding agene product which is therapeutically valuable are inserted into theclonogenic fibroblasts. Gene products of this nature are growth factors,in particular hematopoietic growth factors, and also hormones, forexample insulin, or coagulation factors, for example factor VIII,coagulation inhibitors, enzymes, for example lysosomal enzymes, oradenosine deaminase.

In a particularly preferred embodiment of the present invention,hematopoietic growth factors such as G-CSF or erythropoietin areinserted into the fibroblasts by means of the transfection.

Suitable vectors are required for the transfection. In a preferredembodiment of the present invention, a retroviral vector is used. Inaddition to the requisite essential genes from the retroviral system,this vector can also contain a so-called suicide gene such as thethymidine kinase gene originating from herpes simplex virus. Thisenables cells to be selectively destroyed in the presence ofgancyclovir. In addition, the vectors can contain inducible promoterswhich make it possible to regulate gene expression.

A vector which may preferably be used is the retroviral vector N2, whichis derived from the genome of the Moloney murine leukemia virus (MLV)and contains, as a selective marker, a bacterial gene for resistance toneomycin. The origin of the fragments and the restriction enzymes whichwere used to obtain DNA fragments encoding human interleukin-2, mouseinterferon-y and herpes simplex virus thymidine kinase promoter andother genes, have already been described [Gansbacher et al., J. Exp.Med., 172 (1990) pp. 1217-1224; Gansbacher et al., Cancer Res., 50(1990) pp. 7820-7825 and Gansbacher et al., Blood, 80 (1992) pp.2817-28251].

An additional vector which may preferably be employed in the presentprocess was described in Science, Vol. 256, April 1992, p. 445. This isthe outer envelope of an adenovirus in which the adenoviral genes haveeither been deleted or inactivated in some other way. An antibodypossessing a lysine tail is bonded to the viral envelope with the lysinetail bonding to the transfecting DNA. In this vector system, the DNA isnot packed within the viral envelope but attached externally to theviral envelope. This vector enables relatively large DNA fragments to betransfected.

The human clonogenic fibroblasts which are obtained in accordance withthe novel process may be transfected using the above-described methods.Since only clonogenic fibroblasts are suitable as target cells forstable gene transfection, the fibroblasts which have been obtained inthis way are very suitable for expressing foreign genes.

For example, fibroblasts which have been transfected with cytokine genescan be employed as so-called bystander cells in the context ofvaccinations against tumors or else for the prophylaxis of infections.Fibroblasts which are transfected with other genes can be reintroducedinto the donor and used for supplying the organism over a long periodwith those molecules which are lacking in the relevant disease. In thiscontext, the molecule can, for example, be blood coagulation factorVIII, protein S or protein C, or else insulin, erythropoietin or otherhematopoietic growth factors. It is also possible to provide enzymessuch as glucocerebrosidase or adenosine deaminase.

The novel fibroblasts may be used either in an autologous system or elsein an allologous system, provided this is possible from theimmunological point of view.

According to a preferred embodiment, the genetically transfectedfibroblasts are obtained by means of a physical method of gene transfer.Typical examples of such physical methods of gene transfer areelectroporation, microinjection, particle bombardment and anionic orcationic lipofection.

An electroporation is, for example, carried out as follows: A quantityof 4×10⁶ cells is washed twice with a phosphate-buffered solution ofsodium chloride, [lacuna] in 0.5 ml of an electroporation buffer (20mmol of HEPES, 137 mmol of sodium chloride, 5 mmol of potassiumchloride, 0.7 mmol of Na₂ HPO₄, 6 mmol of sucrose and 1 mg/ml bovineserum albumin, pH 7.0; (Goldstein et al., Nucleic Acids Research, Vol.17 (1989), pages 3959 to 3971)) and incubated on ice for 10 minutes, ina 0.4 cm electroporation cuvette from Bio-Rad, Munich, Germany, togetherwith 20 μg of a DNA. The cells are then electroporated with a capacityof 960 μF in an electroporation device which is charged with 250 V.After a second incubation on ice for 10 minutes, the cells areintroduced into a 50 ml cell culture vessel containing a normal nutrientsolution.

A typical cationic lipofection proceeds, for example, as followsg On theday before the transfection, the cells are applied, at a density of 10⁵per well, to a 35 mm Petri dish, for example from Falcon. In order toobtain the transfection complexes, 2 μg of the DNA, are added, togetherwith different proportions of lipofection agents (DOSPA/DOPE 3:1), whichcan be obtained from Gibco in Germany, to 200 μl of DMEM and the wholeis incubated at room temperature for 30 minutes. The cells are thereuponwashed twice with DMEM and the transfection complexes are added to thecells after having been diluted with DMEM to 1 ml. After an hour, 1 mlof a customary culture medium is added and the medium is changedcompletely 24 hours later.

The genetically transfected fibroblasts which are obtained in accordancewith the invention may be employed as medicaments, in particular forin-vivo treatment. In this context, the genetically transfectedfibroblasts can be used, for example, to mobilize hematopoietic stemcells, insofar as a growth factor gene is present.

The present invention furthermore relates to a support which is made outof a biocompatible material which can preferably be used inendoprostheses and which contains the above-described geneticallytransfected fibroblasts. In this context, the genetically transfectedfibroblasts are first cocultured in vitro with the biocompatiblematerial and the material which has been overgrown in this way is thenimplanted. According to a preferred embodiment of the present invention,the endoprosthesis is a vessel prosthesis which is made out of abiocompatible, preferably human-compatible, synthetic material, forexample fluoropolymers or a polyester, and the genetically transfectedfibroblasts are implanted in vivo using this prosthesis. Alternatively,the endoprosthesis in the form of a vessel prosthesis can consist of abiocompatible, preferably human-compatible material which is derivedfrom natural sources, for example a collagen fleece or a materialobtained from bovine pericardium, with the genetically transfectedfibroblasts being implanted in vivo using this prosthesis.

In a preferred embodiment of the present invention, the fibroblastswhich are obtained in accordance with the novel process are coculturedwith collagen-coated polyvinylpyrrolidone matrices and appliedintraperitoneally (i.p.) or subcutaneously (s.c.) in this form as anorganoid. It is known that such objects are neovascularized in vivo. Thequantity of the gene product which is required in vivo is regulatedthrough the quantity of cells transfected and, where appropriate, alsoby means of the inducible promoter. Instead of the polyvinylpyrrolidonematrices, other suitable support substances such as fluoropolymerfibers, in particular polytetrafluoroethylene fibers, may also be used,which substances are then overlaid with a suitable coating agent such ascollagen. These fibers can then be embedded in an extracellular gelmatrix and applied intraperitoneally, for example, by means of a minorsurgical operation.

EXAMPLE 1

a) Biopsies

Using routine surgical operations, samples of skin and serosa of about0.5-2 cm² in size were obtained from 50 donors who were suffering fromcancer. The biopsy samples were stored in Dulbecco's modified Eagle'smedium (DMEM) and subjected to further processing on the day they wereremoved.

b) Mechanical treatment

The biopsy samples were cut with a scalpel into pieces of less than 2 mmin diameter. These pieces were then plated out on cell culture plateswith the epidermis upwards. DMEM (Gibco BRL) which contained a highcontent of glucose and 10% fetal calf serum (Boehringer Mannheim), andwhich was enriched with L-glutamine and sodium pyruvate, was used as thegrowth medium.

c) Enzymic treatment

After the biopsy samples had been cut into pieces of less than about 0.5cm², these samples were incubated with DMEM medium which containedcollagenase (Biochrom, Berlin), dispase (Boehringer Mannheim) orhyaluronidase (Sigma, Deisenhofen) either individually or in differentcombinations. Cells which were released into the suspension by thistreatment were washed with a phosphate-buffered solution of sodiumchloride and plated out in cell culture bottles at a density of 2×10⁴/cm².

d) Long-term culture

The fibroblast cultures were raised at 37° C. in a moist atmospherecontaining 5% CO₂, with fresh growth medium being added twice a week. Assoon as the cultures reached confluence, the cells were harvested bytreating them with trypsin/EDTA, then washed, counted and sown again ata density of 1×10⁴ cells/cm². The cell counts were extrapolated at eachpassage starting from the number of cells and the dilution factor.

e) Plating efficiency

100 cells were plated out in 10 cm diameter culture plates. On thefollowing day, 5×10⁵ human embryonic WS-1 fibroblasts or NIH3T3 mousefibroblasts were added to each culture plate. The two cell lines, bothof which are obtainable from the ATCC, Rockville, MD, were used asso-called feeder cells after having been irradiated with 100 Gy. After 4weeks of culture, the colonies were stained with 2% methylene blue in50% ethanol and counted, with the medium being changed every 2 weeksduring the culture period.

f) Determination of the cytokines

The supernatants from subconfluent cultures were harvested 24 hoursafter completely changing the nutrient medium. The cells were countedand the cytokine concentrations in the supernatants were determinedusing commercially available ELISA tests.

EXAMPLE 2

The experimental conditions given in Example 1 were employed usingdifferent enzymes (Example 1c) in order to ascertain efficacy forsetting up long-term cultures of diploid fibroblasts. The best resultswere obtained when the biopsy samples, which had been chopped into smallpieces, were first digested with dispase at a concentration of about 2.5U/ml at 4° C. for 16 hours. After this treatment, the epidermis waseasily removed from the cell fragments. The skin cells were then furtherchopped into pieces of a few mm. This cell material was subjected to asecond enzymic treatment, at 37° C. for 3 hours in a shaking water bath,using a mixture of collagenase (200 U/ml) and hyaluronidase (300 U/ml).Finally, the cells were separated by passing them through a 70 μm sieve,washed and transferred to cell culture.

FIG. 1 shows a plot of the donor cells (number) against the number ofdays of culturing, with the number behind/after the curve giving the ageof the human donor. This shows that diploid fibroblast cultures whichwere obtained, using this process, from donors who were younger than 60years of age generally exhibited similar growth characteristics, with amean doubling time of 4.3±0.6 days.

FIG. 2 shows a plot of the donor cells (number) against the number ofdays of culturing for human donors of different ages. In the figure, □denotes preparation of the fibroblasts by enzymic treatment and Δdenotes isolation of individual cells simply by mechanical means. Thisshows that the fibroblast cultures which were prepared using the enzymicprocess clearly had a superior capacity for multiplication as comparedwith those fibroblast cultures which were obtained simply by mechanicaltreatment of the skin biopsy samples.

Using the novel process, diploid fibroblast cultures can be obtained notonly from skin cells but also from peritoneal cells. It was found thatcell cultures which were derived from the serosa initially grew in asimilar manner to the cell cultures derived from the skin, with,however, the diploid fibroblasts originating from the serosa reachingthe plateau at a markedly lower cell count than did the diploid skinfibroblast cultures of a comparable age. FIG. 3 shows a comparison ofthe cell counts plotted against the number of days of culturingfibroblast cultures which originate either from the skin (left-handdiagram) or the serosa (right-hand diagram) of donors aged from 45 to59.

FIG. 4 shows the effect of so-called feeder cells on the platingefficiency of the diploid fibroblast cultures. Diploid skin-cellfibroblast cultures were prepared which were treated enzymically. Theirplating efficiency was determined by sowing the cells at very lowdensity on the cell culture bottles. Whereas the diploid fibroblastswithout feeder cells exhibited hardly any colony formation under theseconditions, it was possible to increase the plating efficiency to 9-24%by adding irradiated human fibroblasts of the WS-1 cell line.

EXAMPLE 3

Particular attention was devoted to the production of cytokines in theautologous diploid fibroblast cultures. For this purpose, culturesupernatants were taken both from non-irradiated diploid fibroblasts andalso from diploid fibroblasts which had been irradiated with 20 or 100Gy. The culture supernatants were taken from the irradiated cells on the3rd and the 8th day after irradiation.

Between 1.2 ng and 20.5 ng of interleukin-6 were found, per 24 hours and10⁶ cells, in the supernatants from non-irradiated diploid fibroblasts.The irradiation did not cause any significant changes in the secretionof IL6. More than one third of the cultures produced measurable amountsof GM-CSF (up to 3 ng/24 hours/10⁶ cells) without significant changeafter irradiation. In contrast with this, the non-irradiated diploidfibroblasts did not produce any measurable quantities of TNFα, but up to50 pg of TNFα were measured per 24 hours and 10⁶ cells in single casesfollowing irradiation. These results are presented in FIG. 5, with thedesignations HP-33, KE-58 and KP-68 indicating different cell lines.

EXAMPLE 4

Preliminary experiments in a mouse model, which are explained in moredetail below, were carried out in order to demonstrate the efficacy ofthe novel process for genetically transfecting human fibroblasts. Theresults from the mouse model can be applied in a corresponding manner tohuman fibroblasts.

a) Vector

The basic retroviral vector N2 is derived from the genome of the Moloneymurine leukemia virus (MLV) and contains the bacterial gene for neomycinresistance as a selection marker. DNA fragments encoding the majorimmediate early human CMV promoter, the adenosine deaminase (ADApromoter), the poly-A signal and mouse GMCSF were cloned into differentsites in the N2 vector. The construct N2 vector/CMV promoter/mouseGM-CSF was prepared by cloning the CMV-GM-CSF fusion product into a XhoI restriction site in the neomycin resistance gene in the N2 vector(FIG. 6, top).

In order to prepare the retroviral vector DC/AD/R/GM-CSF (FIG. 6,bottom), the ADA promoter was cloned in reverse orientation into arestriction cleavage site of the enzyme Mlu I which had been filled inusing the Klenow fragment. The mouse GM-CSF cDNA was likewise cloned inreverse orientation into a cleavage site which had been produced by therestriction enzyme SnaB I, while the poly-A signal was cloned into anApa I site in the 3'LTR polylinker which had been filled in usingKlenow. The retroviral vectors were converted into the correspondingviruses by electroporating the vector DNA into a helper-free ecotropicpackaging cell line (GP±E-86). Following selection on G418 (0.7 mg/mlgenticin, Gibco Laboratory, Grand Island, N.Y.), colonies were isolatedand expanded to form producer cell lines. Cell-free supernatant wastested on NIH 3T3 cells in order to determine the titer of the virus.

FIG. 6 shows the structure of the retroviral vectors which were used.

b) Cell lines and infection of the tumor cells and fibroblasts

CMS5 is a methylcholanthrene-induced, non-metastatic fibrosarcoma from aBALB/c genetic back-ground. NIH 3T3 is a contact-inhibited fibroblastcell line which was established from NIH Swiss mouse embryos (CRL 1658).BALB 3T3 clone A31 are contact-inhibited, non-tumorogenic fibroblastswhich were established from BALB/c mouse embryos (CCL 163). Both thefibroblast cell lines were obtained from ATCC. The cells were culturedin Dulbeccols modified Eagle's medium which was supplemented with 10%fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 2mmol/1 L-glutamine. Virus-producer cell lines, which secreted hightiters of the virus, were employed for infecting the CMS5 cells and thefibroblast cells. Following selection with G418, clones or bulk-infectedcells were expanded into lines and subjected to further analysis.

c) Cytokine determination

Secretion of GM-CSF into the supernatants of retrovirally infected CMS5cells and fibroblasts, and the concentration of GM-CSF in the serum oftreated mice, were determined by a bioassay and confirmed using an ELISAtest. The supernatants from semi-confluent parenteral orGM-CSF-secreting cells were collected after 24 hours, with the cellcount being determined and the supernatant being examined for theproduction of mouse GM-CSF.

10⁶ CMS5 cells which were irradiated with 50 Gy or 10⁶ cells which weretransfected with the vector N2/CMV/GM-CSF/CMS5 were examined. On the dayof irradiation, the cells were plated in small tissue culture bottlesand the quantity of GM-CSF in the 24-hour culture supernatant wasdetermined on the 3rd, 6th, 9th and 12th day following the irradiation.

The biological activity of the GM-CSF to be investigated was determinedby ascertaining the ability of the GM-CSF-containing supernatants toinduce the incorporation of ³ H-thymidine into the DNA ofGM-CSF-de-pendent mouse 32DC13 cells. After the cells had been incubatedovernight without GM-CSF, 10⁴ cells were incubated in each well of a96-well microtiter plate together with dilutions of the test liquids andthe cells were cultured at 37° C. for 6 hours and in 6% CO₂. After that,1 μCi of ³ H-thymidine was added and the incubation was continued at 37°C. for a further 15 hours. After having been washed, the cells wereevaluated using a liquid scintillation counter. The GM-CSF activity wasexpressed as counts per minute (cpm) and the production, in ng per 24hours, by 10⁶ cells was calculated by comparison with a standard curveof recombinant mouse GM-CSF. In addition to this, the production of theGM-CSF was confirmed by means of an ELISA test.

The irradiation of growth factor-producing autologous or allogenic cellsmay be used for preventing further proliferation of the cells followinginjection in vivo. For this reason, GM-CSF-secreting cells were firstirradiated with 50 Gy and the secretion of GM-CSF was then determined upto 12 days after irradiation. It can be seen from FIG. 7 that theproduction of GM-CSF was initially increased following irradiation.Whereas approximately 40 ng/10⁶ cells/24 hours were measured prior toirradiation, the values are approximately 130 ng/10⁶ cells/24 hours onthe 3rd and 6th days after irradiation. On the 9th day, the values fallback to those obtained prior to irradiation, and the production ofGM-CSF/24 hours is approximately one-third of the starting value on the12th day after irradiation. Since the number of surviving cells hasalready fallen to one-tenth of the starting value on the 6th day, it isclear that GM-CSF is still being liberated from cells which are nolonger vital. d) Transfer of the genetically transfected cells into mice

Female BALB/c mice which were 7-10 weeks old were employed for theexperiments. On day 0 and day 2, all the mice were given 150 mg/kgcyclophosphamide, which was administered intraperitoneally. While onegroup of mice were not given any further injections, a second group wasinjected subcutaneously twice on day 3 with 100 ng of recombinantlyprepared mouse GM-CSF. On day 3, the latter group was given a total of10⁷ N2/CMV/GM-CSF-CMS5 cells, which had been irradiated with 50 Gy,which cells were injected subcutaneously at two sites. From the fourthday onwards, blood was transferred daily from the tail veins of the miceinto heparinized Eppendorf tubes. The number of leucocytes wasdetermined in a Neubauer cell chamber after lyzing the erythrocytes.Differential blood pictures were prepared every two days, with thedifferential blood picture being determined after staining withMayGrunwald-Giemsa. Serum values of mouse GM-CSF were determined on days1, 4 and 10 after injecting the irradiated GM-CSF-secreting fibrosarcomacells.

EXAMPLE 5

Female BALB/c mice were treated as described in Example 4d. From the 3rdday onwards, the peripheral leucocyte counts were ascertained daily anda differential blood picture was prepared every 2 days. In these. mousestrains, the basic leucocyte counts are from about 9000 to 10,000leucocytes/μl. As a result of administering cyclophosphamide, theleucocytes declined on the 3rd day down to about 1000 to 1500leucocytes/μl. No major changes in the leucocyte values were evident. upto the 6th day after injecting cyclophosphamide. All the treatmentgroups exhibit approximately the same values. From the 7th day onwards,a difference in the absolute leucocyte count can be seen between theGM-CSF-treated mice and the control mice which were only injected withcyclophosphamide. After subcutaneous injection or single injection ofGM-CSF-secreting autologous cells, the values in the growthfactor-treated animals are, on the 7th and 8th days after beginning thetreatment, approximately twice as high as in the control mice. Thegroups treated with recombinant growth factor, like the animals treatedwith GM-CSF-secreting fibrosarcoma cells, achieve the normal leucocytevalues on the 8th day after the cyclophosphamide injection. By contrast,the control mice are still exhibiting values which are half the normal.FIG. 8 gives the differential blood pictures of the different treatmentgroups as absolute leucocyte subpopulations (monocytes, granulocytes andlymphocytes). In the differential blood picture, approximately 1%monocytes, 15-30% granulocytes and 70-85% lymphocytes are present inuntreated BALB/c mice. On the 5th day after beginning thecyclophosphamide treatment, that is in the phase of absoluteneutropenia, virtually only lymphocytes can be found in the differentialblood pictures in all treatment groups. A clear difference in thedifferential blood pictures can also be observed on the 7th day, whenthe absolute leucocyte values of the mice treated with recombinantGM-CSF and with GM-CSF-secreting cells are approximately twice as highas they are in the animals which have only been treated withcyclophosphamide. The animals which have not been treated with growthfactor have only about 15% of the monocytes and 25% of the granulocytespossessed by the mice which were treated subcutaneously with GM-CSF orwith GM-CSF-secreting fibroblasts and which have already reached normalabsolute granulocyte counts. No differences in the absolute lymphocytevalues can be observed between the different treatment groups.

FIG. 8 consequently demonstrates that even a single injection ofirradiated, genetically transfected autologous cells has biologicaleffects on the blood picture of chemotherapeutically treated mice.

EXAMPLE 6

FIG. 9 depicts the leucocyte count, which changes over the course oftime. The mice were in each case treated with cyclophosphamide. Onecontrol group did not receive any GM-CSF. In a further control group,recombinantly prepared GM-CSF was injected subcutaneously. In the thirdgroup, genetically transfected cells which produced GM-CSF wereadministered subcutaneously.

EXAMPLE 7

Selective destruction of genetically transfected cells in vivo. In thisexample, murine CMS-fibrosarcoma cells containing 2 different constructswere transfected:

1. pCMV.GCSF.iresNEO contains, under the control of the CMV promoter,the human G-CSF gene and, by way of an IRES (internal ribosomal entrysite) the gene for resistance to neomycin. In this case, therefore, theintention is to express G-CSF and neomycin phosphotransferase.

2. pCMV.GCSF.iresTK/NEO contains, under the control of the CMV promoter,the G-CSF gene and also, by way of an IRES, a herpes simplex virusthymidine kinase/neomycin resistance gene fusion. In this case, theintention is to express TK in addition to G-CSF and neomycinphosphotransferase and, as a consequence, to phosphorylate and activategancyclovir after this prodrug has been administered.

FIGS. 10A 10C 10B and 10D shows a plot of the leucocyte count as numbersof cells per nl (n/nl) against time (in days) after subcutaneouslyinocculating 2.5×10⁵ non-irradiated CMS-5 cells of the previouslymentioned construct 1 10A, 10C, 10E, and 10G and of construct 2 10B,10D, 10F, and 10H into the tumors of 3 BALB/c mice. The chronologicalcourse of the leucocyte counts following the tumor inoculations is shownat the bottom left and bottom right of the diagrams, with in this casethe prodrug gancyclovir also having been administered i.p. twice daily.The chronological development (in days) of tumor size (mm) is given forthe same constructs, once again in three BALB-c test mice, in FIGS. 10E,10G, 10F, and 10H.

The two groups of BALB-c mice were in each case injected subcutaneouslywith 2.5×10⁵ non-irradiated CMS-5 cells which had been transfected withthe above-mentioned vectors. From the seventh day (d7) onwards, onegroup of the mice in each case was given gancyclovir (GCV) i.p. twicedaily (see FIG. 10C, 10D, 10G, and 10H). The leucocyte values and thedevelopment of tumors were monitored. The experimental group, which wastreated with GCV and received CMS-5 cells which were transfected withp.CMV.GCSF.iresTK/NEO, exhibits a decline in leucocytes and regressionof the tumor (see FIG. 10D and 10H right). This demonstrates that eventumor cells which have been transfected with the TK gene can beselectively switched off in vivo by means of the "suicide gene"expression mechanism. Side effects were not observed in the mice, whichare also tumor-free at 5 weeks after terminating the GCV administration.

EXAMPLE 8

Reconstitution of hematopoiesis after a single subcutaneous injection offibroblasts which have been transfected with the G-CSF gene.(Transfection of fibroblasts using physical methods of gene transfer, inthis case lipofection, in this case illustrated using the example ofhuman G-CSF).

FIG. 11 shows a plot of leucocyte numbers (WBC/μl) against time afterinjection (in days) following chemotherapy and cytokine therapy in thepresence/absence of transfected fibroblasts. The chronological coursewhen cyclophosphamide alone is administered is shown on the left-handside, while the corresponding chronological course when cyclophosphamideis administered together with the twice-daily subcutaneousadministration of recombinant human G-CSF, termed rhG-CSF for shortbelow, is shown center left. The chronological course of leucocytecounts following administration of cyclophosphamide and a singleinjection of 5×10⁶ G-CSF-gene-transfected BALB-3T3 fibro-blasts can beseen in the center right on the diagram, while the chronological courseof leucocyte counts following the administration of cyclophosphamide anda single injection of 5×10⁶ irradiated G-CSF-gene-transfected BALB/3T3fibroblasts can be seen on the right-hand side. In these plots, thecurves marked with a square, a rhombus and circle represent the changesin the leucocyte contents of the three test mice employed. It is evidentfrom the three diagrams that the regeneration of the leucocytes takesplace at least as rapidly in the groups in which there was a singleinjection of the transfected fibroblasts (center right and right) aswhen there was a twice-daily subcutaneous injection of the recombinantprotein (center left).

The following table shows the effect of G-CSF administration on themobilization of hematopoietic stem cells into the peripheral blood. Thistable compares the day on which all of the mice of the group (in eachcase three mice per group) exhibited at least 3500 leucocyte/μl and thecorresponding values on the following day. In addition, the table givesthe number of colony forming cells, i.e. of cells which provideinformation on the number of hematopoietic stem cells, termed CFU forshort below, and also the values of the CFU to leucocyte ratios in thefour different treatment groups. It can be seen that a single injectionof nonirradiated and also irradiated G-CSF-gene-transfected fibroblastsmakes possible a mobilization of hematopoietic stem cells into theperipheral blood which is comparable to that obtained with thetwicedaily subcutaneous injection of rhG-CSF. This is of clinicalinterest since, in the case of the stem cell transplantation which isemployed following high-dose chemotherapy in patients possessing avariety of tumors, these stem cells have to be collected, for example,by means of leukophoresis. Following low-dose chemotherapy, the patientsare currently given G-CSF subcutaneously over several days (as discussedin connection with the above animal model) for this purpose.

However, this form of parenteral administration is not only technicallyelaborate but also much more expensive. It would be desirable,therefore, to replace the frequent subcutaneous injections with a singleinjection.

    __________________________________________________________________________    Effect of G-CSF administration on mobilization of hematopoietic stem          cells into the peripheral blood.                                                       Day x              Day x + 1                                                  Leucocytes                                                                          CFU/μl                                                                          CFU/Leucocytes                                                                        Leucocytes                                                                           CFU/μl                                                                           CFU/Leucocytes                       __________________________________________________________________________    Cyclophosphamide                                                                       5533 ± 348                                                                       18 ± 3.8                                                                        0.333 ± 0.109                                                                      6067 ± 354                                                                         4 ± 1.3                                                                         0.060 ± 0.016                     + G-CSF s.c.                                                                           6500 ± 1021                                                                      35 ± 4.5                                                                        0.600 ± 0.081                                                                      16767 ± 1573                                                                       66 ± 13.9                                                                       0.400 ± 0.047                     + G-CSF/BALB                                                                           4183 ± 261                                                                       10 ± 4.7                                                                        0.200 ± 0.081                                                                      9700 ± 1510                                                                       46 ± 9.6                                                                         0.467 ± 0.072                     s.c.                                                                          + irradiated                                                                           7483 ± 1204                                                                      29 ± 1.9                                                                        0.400 ± 0.047                                                                      12800 ± 1746                                                                      90 ± 8.0                                                                         0.733 ± 0.072                     G-CSF/BALB s.c.                                                               __________________________________________________________________________     The ± values mentioned above for leucocytes, CFU and CFU following the     value are the corresponding standard errors.                                  The following were administered to the mice:                                  cyclophosphamide: d0 + d2 (i.p. 150 mg/kg)                                    + GCSF: cyclophosphamide + rhGCSF d3-d7 (s.c. 125 μg/kg, 2 ×         daily)                                                                        + GCSF/BALB: cyclophosphamide + hGCSF-gene-lipofected BALB 3T3, d3 (s.c.      × 10.sup.6 cells)                                                       + irradiated GCSF/BALB: cyclophosphamide + irradiated (50 Gy)                 hGCSF-gene-lipofected BALB 3T3, d3 (s.c., 5 × 10.sup.6 cells).     

We claim:
 1. A process for producing transfected fibroblasts,comprising:(a) removing a fibroblast containing tissue sample from asubject, (b) preparing a suspension of single cells of said tissuesample, (c) washing said suspension, (d) culturing the washed cells inthe presence of feeder cells selected from the group consisting ofirradiated human fibroblasts and irradiated murine fibroblasts, underconditions favoring proliferation of fibroblasts from said sample toproduce clonogenic fibroblasts, and (e) inserting at least one gene intosaid clonogenic fibroblasts wherein said gene encodes a biologicallyactive protein.
 2. The process of claim 1, wherein said biologicallyactive protein is a therapeutically active protein.
 3. The process ofclaim 2, wherein said therapeutically active protein is selected fromthe goup consisting of a growth factor, a hormone, an enzyme, acoagulation factor or a coagulation inhibitor.
 4. The process of claim3, wherein said growth factor is a hematopoietic growth factor.
 5. Theprocess of claim 1, further comprising transfecting said fibroblastswith a suicide gene.
 6. The process of claim 5, wherein said suicidegene is a herpes simplex virus thymidine kinase gene.
 7. The process ofclaim 1, wherein said gene is operably linked to a promoter.
 8. Theprocess of claim 1, wherein said gene is inserted byamethodselectedfromthegroup consisting of electroporation,microinjection, particle bombardment and lipofection.
 9. The process ofclaim 1, wherein said fibroblasts are human fibroblasts.
 10. The processof claim 1, wherein said feeder cells are allogeneic to a subject fromwhich they are taken.
 11. The process of claim 1, further comprisingcontacting said tissue sample with at least one enzyme selected from thegroup consisting of collagenase, dispase, and hyaluronidase.
 12. Theprocess of claim 7 wherein said promoter is an inducible promoter. 13.The process of claim 1 wherein said fibroblast containing tissue is abiopsy from a patient having cancer.
 14. The method of claim 1 furthercomprising (f) isolating and expanding a transfected cell of step (e).15. A process for producing clonogenic fibroblasts, comprising:(a)removing a fibroblast containing tissue sample from a subject, (b)preparing a suspension of single cells of said tissue sample, (c)washing said suspension, (d) culturing the washed cells in the presenceof feeder cells selected from the group consisting of irradiated humanfibroblasts and irradiated murie fibroblasts, under conditions favoringproliferation Qf fibroblasts from said sample to produce clonogenicfibroblasts, and (e) isolating and then expanding the isolatedclonogenic fibroblasts.
 16. A method for mobilizing hematopoietic cellsin a subject comprising administering to a subject in need thereof anamount of a therapeutic composition comprising transfected fibroblastsproduced by the method of claim 7 and a pharmaceutically acceptablecarrier wherein said biologically active protein is granulocytemacrophage colony stimulating factor (GM-CSF) and wherein said amount issufficient to mobilize hematopoietic cells.
 17. The method of claim 16wherein administering the transfected fibroblasts comprises the step ofimplanting a vessel prosthesis comprising a biocompatible materialcontaining the transfected fibroblasts into said subject.
 18. The methodof claims 16 wherein said transfected fibroblasts are irradiated. 19.The method of claim 18 wherein administering the transfected fibroblastscomprises the step of implanting a vessel prosthesis comprising abiocompatible material containing the transfected fibroblasts into saidsubject.
 20. A method for mobilizing hematopoietic cells in a subjectcomprising administering to a subject in need thereof an amount of atherapeutic composition comprising transfected fibroblasts produced bythe method of claim 7 and a pharmaceutically acceptable carrier whereinsaid biologically active protein is granulocyte colony stimulatingfactor (G-CSF) and wherein said amount is sufficient to mobilizehematopoietic cells.
 21. The method of claim 20 wherein said transfectedfibroblasts are irradiated.
 22. The method of claim 20 whereinadministering the transfected fibroblasts comprises the step ofimplanting a vessel prosthesis comprising a biocompatible materialcontaining the transfected fibroblasts into said subject.
 23. The methodof claim 21 wherein administering the transfected fibroblasts comprisesthe step of implanting a vessel prosthesis comprising a biocompatiblematerial containing the transfected fibroblasts into said subject.